To prepare asymmetric LUVs with POPE (30%): POPS (30%): cholesterol (40%) in the inner leaflet and POPC (60%): cholesterol (40%) in the outer leaflet (all lipid compositions are mol% unless otherwise noted), 8 mM lipid acceptor LUVs (POPE (30%): POPS (30%): cholesterol (40%)) were prepared as above with 20 mM Tris 100 mM CsCl, or 20 mM Tris 100 mM NaCl 25% (w/w) sucrose at pH 7.4. 16 mM donor lipid MLVs (100% POPC) were prepared using 20 mM Tris 100 mM NaCl buffer with 40 mM MαCD. 1 ml acceptor LUVs were diluted with 20 mM Tris 100 mM NaCl and centrifuged to remove CsCl or sucrose (189,000 g, 37,500 rpm, 45 min, Beckman L8–80M, SW-60 rotor). The acceptor LUVs pellet was resuspended in 1 ml 20 mM Tris 100 mM NaCl pH 7.4 buffer. Donor MLVs were incubated in a 55°C shaker for 2 hours and then mixed with resuspended acceptor LUVs. The 1 ml acceptor and 1 ml donor mixture was incubated in a 37°C shaker for 45 min. CsCl-containing acceptor/donor mixture was loaded on top of a 7% (w/w) sucrose solution and sucrose-containing acceptor/donor mixture was loaded on top of a 9% (w/w) sucrose solution. Acceptor and donor were separated by using centrifugation (189,000 g, 37,500 rpm, 45 min, Beckman L8–80M, SW-60 rotor). After centrifugation, the pellet was resuspended in 20 mM Tris and 100 mM NaCl pH 7.4 buffer. Lipid concentration was determined by using thin layer chromatography (TLC).
Sw60 rotor
Manufactured by Beckman Coulter
Sourced in United States
The SW60 rotor is a fixed-angle ultracentrifuge rotor designed for Beckman Coulter ultracentrifuge systems. It is capable of achieving a maximum speed of 60,000 revolutions per minute (rpm) and a maximum relative centrifugal force (RCF) of 326,000 x g. The SW60 rotor is suitable for a variety of applications that require high-speed centrifugation, such as the separation and purification of macromolecules, organelles, and other subcellular components.
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Lab products found in correlation
Rnasin, by Promega (3 mentions)
Ua 6 gradient fractionator, by Teledyne (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Gradient master 107, by BioComp Instruments (2 mentions)
Sw32ti rotor, by Beckman Coulter (2 mentions)
Xl 90, by Beckman Coulter (2 mentions)
Optiprep, by Merck Group (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Tla45 rotor, by Beckman Coulter (1 mentions)
Js13.1 rotor, by Beckman Coulter (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Optiprep, by Axis-Shield (1 mentions)
Oriole, by Bio-Rad (1 mentions)
Flag m2 bead, by Merck Group (1 mentions)
Immobilon transfer membrane, by Merck Group (1 mentions)
Optiprep, by Abbott (1 mentions)
51 protocols using sw60 rotor
1
Asymmetric Liposome Preparation and Separation
2
RIG-I Oligomerization and Virus Interaction
3
Tobacco Pollen Fractionation and Analysis
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Nicotiana tabacum (L.) pollen collected in the Botanical Garden Città Studi, as described above, was hydrated in a humid chamber overnight. Pollen (3 mg/mL) was germinated in BK medium as reported above, with or without squalestatin 1 μM or myriocin 5 μM. Pollen tubes were rinsed with 10 mL of incomplete TNE buffer (mM Tris, 150 mM NaCl, mM EGTA, 1 mM PMSF, 10 μg/mL TAME) containing 12% sucrose, with or without squalestatin 1 μM or myriocin 5 μM and centrifuged at 2000 r.p.m. for 10 min at 10 °C in a Beckmann JS13.1 rotor. Pollen tubes were homogenized on ice in two volumes of complete TNE (mM Tris, 150 mM NaCl, mM EGTA, 1 mM PMSF, 10 μg/mL TAME, 10 μg/mL leupeptin, 10 μg/mL pepstatin A, 4 μM aprotinin, 8 μM antipain) using a 2 mL Potter (teflon/glass) homogenizer. The homogenate was centrifuged at 572 g for 4 min at 4 °C and the post nuclear supernatant loaded onto a 20% sucrose cushion (3 mL) in incomplete TNE buffer and centrifuged at 64,200 g (Beckman SW-60 rotor) for 30 min at 4 °C. The P2 pellet was resuspended in cold, complete PEM buffer. Aliquots of P2 and supernatant (S2) were protein-assayed (Bradford) using BSA as standard protein.
4
Polysome Profiling of Procyclic Trypanosomes
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3–5×108 procyclic cells were treated with cycloheximide (100μg/ml) for 5 minutes, harvested at room temperature by centrifugation (850g, 8min, 20°C), washed once in 1ml of ice-cold PBS and lysed in 300μl of lysis buffer (20mM Tris pH7.5, 20mM KCl, 2mM MgCl2, 1mM DTT, 1200u RNasin (Promega), 10μg/ml leupeptin, 100μg/ml cycloheximide, 0.2% (vol/vol) IGEPAL) by passing 20–30 times through a 21G needle. After pelleting insoluble debris by centrifugation (17000g, 10min, 4°C) and adjusting to 120mM KCl, the clarified lysate was layered onto a 17.5–50% sucrose gradient (4ml) and centrifuged at 4°C for 2 hours at 40000 rpm in Beckman SW60 rotor. Monitoring of absorbance profiles at 254nm and gradients fractionation was done with a Teledyne Isco Foxy Jr. system. RNAs from pooled fractions were purified using TriFast. To control for the efficiency of RNA isolation, equal amounts of a human β-globin in vitro transcript were sometimes added to each of the collected fractions before RNA purification.
5
Influenza B Virus Purification Protocol
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Madin-Darby Canine Kidney (MDCK) cells were grown in Dulbecco’s Modified Eagles Medium (DMEM, GIBCO,11995-040) containing 10% fetal bovine serum (FBS, GIBCO, Grand Island, NY, USA) and a penicillin–streptomycin mix (100 units/mL of penicillin and 100 μg/mL of streptomycin). Influenza B viruses B/Brisbane/60/2008 (Victoria lineage) and B/Florida/4/2006 (Yamagata lineage) obtained from BEI Resources were grown in MDCK cell at 37 °C for 48–72 h.
Virus was cultured in DMEM containing 1ug/mL TPCK-trypsin and 0.2% Albumin from bovine serum (BSA, GIBCO, Grand Island, NY, USA ). Viruses were inactivated by 0.04% formaldehyde and collected through ultracentrifugation from the supernatant of culture solution. The viruses were then added upon the 30% sucrose cushion and ultracentrifugation at 22,500× g for 2 h in 4 °C using a Beckman SW60 rotor. Finally, the supernatant was discarded and virus particles were resuspended in PBS.
Virus was cultured in DMEM containing 1ug/mL TPCK-trypsin and 0.2% Albumin from bovine serum (BSA, GIBCO, Grand Island, NY, USA ). Viruses were inactivated by 0.04% formaldehyde and collected through ultracentrifugation from the supernatant of culture solution. The viruses were then added upon the 30% sucrose cushion and ultracentrifugation at 22,500× g for 2 h in 4 °C using a Beckman SW60 rotor. Finally, the supernatant was discarded and virus particles were resuspended in PBS.
6
Iodixanol Gradient Floatation for Exosome Isolation
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We carried out a discontinuous iodixanol gradient floatation as described in (69 ). Briefly, solutions of 40, 30, 10% iodixanol were made by mixing appropriate amounts of a homogenization buffer (0.25 M sucrose, 1 mM EDTA, 10 mM Tris-HCl pH 7.4) and an iodixanol working solution (0.25 M sucrose, 6 mM EDTA, 60 mM Tris pH 7.4 plus stock solution of Optiprep 60% (w/v), (Axis-Shield PoC, Norway). Exosomes isolated with the ultracentrifugation-based method were resuspended in PBS (260 μl) and added to 1 ml of 60% Optiprep and placed at the bottom of a polyallomer tube. The gradient was formed by layering 0.5 ml of 40%, 0.5 ml of 30%, 1.8 ml of 10% solutions on top of each other and centrifuged for 17 h at 192,000 × g in a SW60 rotor (Beckman Coulter, Cassina de’ Pecchi, MI, Italy). Thirteen fractions (330 μl) were collected from the top of the tube. The refractive index of each fraction was assessed with a refractometer (Carl Zeiss, Oberkochen, Germany), and the relative density was calculated using the linear relationship between the refractive index and the density (70 (link)).
7
Purification of Archaeal Virus APBV1
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APBV16 (link) was propagated in A. pernix strain K117 (link). Virions were collected from cell-free supernatant by precipitation with polyethylene glycol and purified by CsCl density gradient centrifugation in a Beckman SW60 rotor at 48,000 rpm for 24 h. The virion-containing fractions were collected and dialyzed against distilled water.
8
Evaluating sHsps Assembly Formation
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Luciferase (1 μM) was denatured at 44°C for 10 min in buffer D in the presence of 5 μM sHsps (1.66 μM IbpAEc and 3,34 μM IbpBEc in case of IbpABEc). To verify sHsps ability to form assemblies, 150 μl of each sample was applied on a 3.6 ml 10–60% glycerol gradient in buffer E (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 20 mM Mg acetate, 2 mM DTT). Samples were then centrifuged at 10°C in a Beckman SW 60 rotor at 160,000 g for 1 h, fractions were collected from the top. Protein distribution in each fraction was verified by SDS–PAGE followed by Oriole (Bio-Rad) fluorescent staining.
9
Polysome Profiling of Cycloheximide-Treated Cells
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107 cells were treated with cycloheximide (100 μg/ml) for 5 minutes washed once in cold PBS/cycloheximide (100 μg/ml) and lysed in 400 μl buffer (20 mM Tris–HCl, pH 7.4, 5 mM MgCl2, 150 mM NaCl, 1% Triton X‐100, 100 μg/ml cycloheximide, 1× complete protease inhibitors (Roche)). The lysates were centrifuged at 9,300 g for 10 min at 4°C, and the supernatants were applied to linear 17.5–50% sucrose gradients in 20 mM Tris–HCl (pH 7.4), 5 mM MgCl2, and 150 mM NaCl. Centrifugation was carried out at 35,000 rpm for 2.5 h at 4°C in a Beckmann SW60 rotor. Gradients were eluted with an ISCO UA‐6 gradient fractionator, and polysome profiles were recorded by continuously monitoring the absorbance at 254 nm. In order to calculate the fraction of ribosomes engaged in translation, the area under the polysomal part of the curve was divided by the area below the entire curve.
10
Polysome Profile Analysis
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Cells grown in 10-cm dishes were washed once with ice-cold PBS containing 100 µg/ml cycloheximide (CHX) and directly lysed using 290 µl of polysome lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 µg/ml CHX, 1% Triton X-100, 40 U/ml RNasin (Promega) supplemented with EDTA-free complete protease inhibitors (Roche)), scraped and harvested into microtubes. Lysates were rotated end-over-end at 4°C for 10 min and centrifuged at 10,000 × g for 10 min at 4°C to remove nuclei and cell debris. The concentration of lysates was adjusted by measuring the absorbance at 260 nm, and equal amounts (250 µl) were loaded on top of 3.95 ml 17.5–50% sucrose density gradients, for which sucrose was dissolved in a buffer containing 20 mM Tris-HCl, pH 7.5, 5 mM MgCl2, and 150 mM NaCl. Gradients were subjected to ultracentrifugation at 40,000 rpm using a SW60 rotor (Beckman) for 105 min at 4°C. Fractions were eluted from the top of the gradient and polysome profiles were recorded by continuously measuring the absorbance at 254 nm using a Teledyne ISCO Foxy RI system in combination with PeakTrak software. To quantify the percentage of monosomal and polysomal ribosomes, the area under the curve corresponding to monosomal, polysomal and total ribosomes was integrated.
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